Letters
DOI: 10.1038/s41477-017-0024-6
Endogenous miRNA in the green alga
Chlamydomonas regulates gene expression
through CDS-targeting
Betty Y-W. Chung 1*, Michael J. Deery2, Arnoud J. Groen2, Julie Howard2 and David C. Baulcombe 1*
MicroRNAs (miRNAs) are 2124-nucleotide RNAs present in
many eukaryotes that regulate gene expression as part of the
RNA-induced silencing complex. The sequence identity of the
miRNA provides the specificity to guide the silencing effec-
tor Argonaute (AGO) protein to target mRNAs via a base-
pairing process1. The AGO complex promotes translation
repression and/or accelerated decay of this target mRNA2.
There is overwhelming evidence both in vivo and in vitro that
translation repression plays a major role37. However, there
has been controversy about which of these three mechanisms
is more significant in vivo, especially when effects of miRNA
on endogenous genes cannot be faithfully represented by
reporter systems in which, at least in metazoans, the observed
repression vastly exceeds that typically observed for endog-
enous mRNAs8,9. Here, we provide a comprehensive global
analysis of the evolutionarily distant unicellular green alga
Chlamydomonas reinhardtii to quantify the effects of miRNA
on protein synthesis and RNA abundance. We show that,
similar to metazoan steady-state systems, endogenous miRNAs
in Chlamydomonas can regulate gene expression both by
destabilization of the mRNA and by translational repression.
However, unlike metazoan miRNA where target site utilization
localizes mainly to 3UTRs, in Chlamydomonas utilized target
sites lie predominantly within coding regions. These results
demonstrate the evolutionarily conserved mode of action for
miRNAs, but details of the mechanism diverge between the
plant and metazoan kingdoms.
Recent in vivo studies in mammalian cells provide support for
endogenous messenger RNA destabilization over translation repres-
sion as the dominant effect of miRNA under steady-state conditions9.
However, an inducible zebrafish embryo system in which miR430 is
expressed only two hours post fertilization reveals that translation
repression occurs prior to accelerated mRNA decay10. This conclu-
sion was further supported by findings in mouse liver, primary mac-
rophages, and primary B and T cells8; through a reporter system in
human HeLa cell lines11; and Drosophila S2 cells12.
In contrast to the metazoan systems, there is a lack of compre-
hensive studies on the endogenous effects of miRNAs in plants and
the question remains as to whether miRNA modulates by translation
repression and/or promoting mRNA turnover. In plants, miRNA-
mediated gene regulation does occur1315, but, unlike metazoan sys-
tems, the targets can be in the coding sequence as well as 3UTR and
the mechanism may involve endonucleolytic cleavage rather than
accelerated decay or translation inhibition16,17. Most plant studies,
however, are based on individual miRNAs or reporter assays that
epartment of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK. 2Cambridge System Biology Centre and Department of Biochemistry,
D
1
University of Cambridge, Cambridge CB2 1GA, UK. *e-mail: bcy23@cam.ac.uk; dcb40@cam.ac.uk
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may not be informative about endogenous mRNA systems9,18,19. We
therefore utilized the unicellular green alga Chlamydomonas rein-
hardtii, for which we have previously discovered and characterized
its miRNAs20 and generated DCL3 mutants21.
Chlamydomonas is a particularly amenable experimental sys-
tem because its unicellularity reduces complications with tissue-
specific effects. Similar to higher plants, the machinery for
miRNA-mediated translation regulation is also functional in
Chlamydomonas, where the seed-region rule utilized by the meta-
zoan system is adequate for translation repression, at least within
reporter systems22. In this present study, we utilized two silenc-
ing mutants raised from our previous forward genetic screen at
dcl321 and ago3 (B.Y.-W.C. et al. manuscript in preparation). The
dcl3-1 mutant results in almost complete loss of miRNA as well as
21-nucleotide small interfering (si)RNAs whereas ago3-25 is defec-
tive in AGO3 that binds to miRNA and is required for translation
repression in the reporter system23. Neither mutant had obvious
growth differences or morphological abnormalities under normal
conditions21. Any effect seen in both dcl3-1 and ago3-25 on gene
expression is likely, therefore, to be direct rather than an indi-
rect secondary consequence of metabolic changes due to loss of
miRNA-mediated regulation.
Here, through a combination of ribosome profiling, parallel RNA
sequencing (RNA-Seq), small RNA (sRNA)-Seq and quantitative
proteomics at mid-log phase of the dcl3-1 mutant, its correspond-
ing complemented strainandtheago3-25mutant, we have demon-
strated that, in contrast to the metazoan system, the primary effect
of miRNA in Chlamydomonas is through interaction with CDS
regions instead of 3UTRs. However, similar to the metazoan sys-
tem, miRNA in Chlamydomonas reinhardtii can also modulate gene
expression via means of translational repression and mRNA turn-
over. Finally, and perhaps the most striking observation, is that the
translation apparatus itself is differentially regulated at the level of
translation efficiency but not RNA abundance in the presence of the
miRNA machinery.
DCL3 and global gene expression
To explore the possibility that DCL3-dependent miRNA or siRNA
regulates gene expression by either promoting mRNA turnover or
through interfering with translation, we applied ribosome profiling,
parallel RNA-Seq and quantitative 15N proteomics to biological
triplicates of the vegetative mid-log phase dcl3-1 mutant and its
corresponding complemented derivative (abbreviated as Cdcl3)
carrying a wild-type DCL3 allele introduced into the mutant strain.
The experimental protocol is summarized in Supplementary Fig.1,
Letters NATURe PlAnTs

and Supplementary Fig.2 illustrates the high degree of reproduci
bility between biological repeats in these data.
The slightly smaller footprint size of plant/algae ribosomes leads
to differences in the phasing patterns compared with mammalian
ribosome profiling studies24. In both the complemented strain Cdcl3
and the dcl3-1 mutant, the 5end of the 27-nucleotide ribosome-
protected fragments (RPFs) mapped predominantly to the second
codon position; in contrast and, as expected, RNA-Seq reads were
uniformly distributed at all three codon positions (Fig.1a,b). The
RPF 5end position distributions at start and stop codons were also
similar in the dcl3-1 and Cdcl3 strains (Fig.1c,d respectively) in that
there was a sharp 27-nucleotide peak on the start codon (reflect-
ing the rate-limiting initiation step of translation) and a sharp
28-nucleotide peak on the stop codon (reflecting the conformation
change from an elongating ribosome to a terminating ribosome;
Supplementary Fig. 3B)24. In contrast, the RNA-seq reads are not
limited to coding regions (Fig.1e,f and Supplementary Fig.3B).
The validity of these data was further confirmed with the analy-
sis of DCL3. There were multiple DCL3 mRNA reads from three
replicate samples of the Cdcl3 strain that were restricted to the open
reading frame (ORF) in the RPF data sets. In dcl3-1, the reads were
from the region on the 5side of the mutagenic DCL3 insertion
(Supplementary Fig.3C). Finally, RPFs, RNA abundance (RA) and
translational efficiencies (TEs) for expressed genes are well corre-
lated between dcl3-1 and Cdcl3 (R2=0.95, 0.97 and 0.98 for TE, RPF
and RNA, respectively, Supplementary Fig.3E). From these data, we
conclude that any global effect of DCL3 on the translatome is minor
but we could not rule out quantitative effects on a subset of RNAs.
To explore this possibility, we refined our analysis by dividing
the mRNA profiles into those with or without predicted targets of
the DCL3-dependent miRNAs. The first stage in this analysis was
to re-evaluate the miRNA precursors in C. reinhardtii that we had
previously identified as being both coding and non-coding RNAs.
Now, however, with the use of the RPF data to identify translated
ORFs, we find that all miRNAs in this alga derive from introns or
the exons (3UTR or coding) of mRNAs. Supplementary Fig.4 and
Table2 show an updated summary of the 42 miRNA precursors in
C. reinhardtii described in Valli et al. 201621.
Our subsequent analysis differentiated mRNAs with miRNA
targets in the 5UTR, CDS and 3UTR from those without targets.
The CDS regions were defined by the R software Bioconductor
package riboSeqR that utilizes the triplet periodicity of ribo-
some profiling for the de novo inference of AUG-initiated cod-
ing sequences that are supported by RPFs24 and we used the
seed-sequence rule to identify miRNA target motifs25,26. This rule
requires base-pairing of the first eight nucleotides of miRNA and
it is supported by direct assay of miRNA targeting and structural
studies of human AGO227 and by experimental tests in higher
plants28 and C. reinhardtii22.
To identify the miRNA-target mRNAs, we first filtered for the
19 most abundant DCL3-dependent miRNAs in our sRNA-Seq
data (Supplementary Fig. 5; see also Methods). We then applied
the TargetScan prediction algorithm25,26 to the mRNAs with RPF-
validated ORFs. This criterion meant that the TargetScan algorithm
was applied to 13,073 expressed transcripts (out of 17,741 annotated
transcripts) of which 2,439 do not contain any predicted octonucle-
otide miRNA target sites. Of all the predicted target sites, a larger
proportion (70%) are located in the CDS (Fig.2a) compared with
UTRs (10% for 5UTR and 36% for 3UTR). This distribution is
probably, at least in part, a reflection of the greater length of the

a b
Relative proportion

Relative proportion

25 26 27 28 29 30 31 32 33 34 35 25 26 27 28 29 30 31 32 33 34 35
Read length (nt) Read length (nt)
c d
Relative read density

Relative read density

100 Start 100 200 300 400 200 100 Stop 100 Start 100 200 300 400 200 100 Stop
Location of 5 end of reads relative to start and stop codons Location of 5 end of reads relative to start and stop codons
e f
Relative read density

Relative read density

100 Start 100 200 300 400 200 100 Stop 100 Start 100 200 300 400 200 100 Stop
Location of 5 end of reads relative to start and stop codons Location of 5 end of reads relative to start and stop codons

Fig. 1 | Ribosome profiling data. a,b, Mapping the 5ends of ribosome-protected fragments (RPFs) and corresponding RNA-Seq respectively, as a function
of read size class (nucleotides), within nucleus-encoded coding ORFs. Red, green and blue bars indicate the proportion of reads that map to codon
positions 0, 1 and 2 (respectively). c,d, 5end positions of 27-nucleotide RPFs relative to start and stop codons (nucleotides). Reads were derived from
strain Cdcl3 and dcl3-1 (respectively) and summed over all transcripts. Phasing is indicated using the same colours as in a and b. e,f, 5end positions of
all RNA-Seq reads relative to start and stop codons (nucleotides). Reads were derived from strain Cdcl3 and dcl3-1 (respectively) and summed over all
transcripts. Phasing is indicated using the same colours as in a and b.

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NATURe PlAnTs Letters
1,354
a
292
5 UTR CDS 3 UTR
668 249
145 9,108
4,746

1,262 3,090 5,205

Non-target = 2,439

b Target sites

Number of mRNAs 1,354 9,108 4,746 292 5,205 1,262

Number with high
complementarity 481 25,047 3,849 63 4,523 604
Target sites
Number of octonucleotide 1,692 58,881 8,849 253 10,313 1,545
target sites
50.0
Percentage of octonucleotide sites
with high complementarity

37.5

25.0

12.5

0
All sites Region exclusive

Fig. 2 | Distribution of octonucleotide target sites. a, Venn diagram showing the number of transcripts predicted to be targeted with the octonucleotide
rule. b, Proportion of octonucleotide target sites that also have at least 50% complementarity from nucleotides 1121 of the miRNA.

CDS compared with UTR regions. Using a more stringent miRNA
targeting rule did not have a large effect on these numbers: about
half of the mRNA seed sequence targets also have >50% sequence
complementarity to the relevant miRNA in the sequences upstream
of the 3eight nucleotides (Fig.2b).
Next, we excluded the RNAs with predicted target sites in more
than one region (5 UTR/CDS/3UTR) because for these it would
have not been possible to differentiate the effects of miRNA act-
ing in the different regions. In addition, we also excluded mRNAs
with miRNA precursors because they are unstable in the presence of
DCL3 as a consequence of miRNA processing (see Supplementary
Fig.4 and ref. 21). Following application of these filters, our further
analysis was based on 292 mRNAs with 5UTR targets, 5,205 with
CDS targets, 1,262 with targets in the 3UTR and the 2,439 without
predicted targets.
Similarly to previous studies810, we plotted cumulative distribu-
tions of differential translation efficiency, total RPF and RA for tar-
get and non-target mRNAs in the dcl3-1 mutant and Cdcl3 to assess
the miRNA-mediated effects of DCL3 (Fig. 3a,b). Differential TE
is computed as (RPFCdcl3/RNACdcl3)/(RPFdcl3-1/RNAdcl3-1). The analysis
revealed that, similar to the analysis of zebrafish10, the major effects
of Dicer loss of function (dcl3-1 versus Cdcl3) were on mRNAs con-
taining target sites within the CDS and the effect is more significant
in the RPF than the RNA data, contributing to its significant but
small effect in TE. The effects were evident as a shift to increased
RNA abundance for mRNAs with target sites in dcl3-1 and they
are consistent with the canonical role of miRNAs as negative
regulators.
The difference in dcl3-1 versus Cdcl3 was greater in transcripts
with CDS rather than UTR target sites and this effect appears to be
dosage dependent, where mRNAs with four or more CDS targets
were affected to a greater extent than those with fewer target sites
(Fig.3c). However, this dosage-dependent effect was not observed
for mRNAs containing target sites in the UTRs (Supplementary
Fig.6A). Furthermore, these effects are also consistent at the protein
level for mRNAs with supportive proteomics data (Supplementary
Fig.6B).
As the key AGO in Chlamydomonas known to be associated
with miRNA is AGO3, which mediates translational repression in
a reporter system23, we also performed ribosome profiling as well as
corresponding RNA-Seq on an AGO3 mutant (ago3-25), raised from
the same forward genetic screen as dcl3-121, as well as the corre-
sponding parental strain and the wild type CC-1883 (B.Y.-W.C. et al.,
manuscript in preparation) to further validate whether these effects
are truly due to the miRNA machinery. In support of this, we also
observed the dosage-dependent effect only for mRNAs contain-
ing target sites within the CDS in the ago3-25 mutant background
(Fig.3d and Supplementary Fig.6A).
The global effect of mRNA repression is not likely to be due to target
RNA cleavage as there are only 85 potential CDS target sites (83 mRNAs)

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Letters NATURe PlAnTs

a e
1.0 1.0 1.0 100
Cumulative distribution

0.8 0.8 0.8

0.6 0.6 0.6

Relative miRNA abundance (%)

miR-C89
0.4 0.4 0.4 75
0.2 0.2 0.2
0.0 0.0 0.0
1.0 0.5 0.0 0.5 1.0 1.0 0.5 0.0 0.5 1.0 1.0 0.5 0.0 0.5 1.0 50 miR-C7085
b TE RPF RNA miR-C112
0.07
5.4 108 miR-C20399
Area difference

25
0.04 7.4 106 miR-9897
miR-1157
0.01 0.96
0.01 0.74 0.22 0.40 0.54 0.63
0.00
0
0.02 Biorep 1 Biorep 2 Biorep 3
5 UTR CDS 3 UTR
c dcl3-1 vs Cdcl3 f
0.30

Cumulative distribution
1.0 1.0 1.0
0.8 0.8 0.8
Area difference

0.22
0.6 0.6 0.6
16
0.14 1.1 10 0.4 0.4 0.4
12
4.1 10
6 105 0.2 0.2 0.2
0.0027
0.05 0.0047 0.0051 0.13
0.39 0.084 0.24 0.85 0.90 0.0 0.0 0.0
0.00
0.03

.0
.5

0
5
1.0

.0
.5

0
5
1.0

.0
.5

0
5
1.0
0.

0.

0.
0.

0.

0.
0

0
1

1
d TE RPF RNA
ago3-25 vs Parent
0.30 5.00
0
3.75
log10(P value)

0.22
Area difference

0.14 2.50
4.8 107
4.4 107 4.1 107
0.05 5
6.5 104
1.25
2.5 10
0.65 0.75 0.04 0.99 0.46
0.00
0.03 0.00
t=1 t=2 t=3 t4 CDS 3 UTR

Fig. 3 | miRNA downregulates gene expression primarily through mRNA destabilization by CDS targeting. a, Cumulative distributions of TE (left),
RPF (middle) and RA (right) log2 fold changes in dcl3-1 relative to Cdcl3. Colours correspond to genes containing predicted octonucleotide miRNA
target sites exclusively in the 5UTR (orange), CDS (green) or 3UTR (blue), or no targets (black). b, Bar graph of differences between area under
cumulative distribution of mRNA containing target sites and non-target-containing mRNAs (5UTR, CDS and 3UTR in orange, green and blue,
respectively). Significance (KolmogorovSmirnov test) of the differences is indicated above each bar; p values less than or equal to 0.01 are highlighted in
red. c,d, Bar graph of differences between area under cumulative distribution of mRNA containing 1 (red), 2 (blue), 3 (purple) or 4 or more (green)
CDS-exclusive target sites and non-target-containing mRNAs. Significance (KolmogorovSmirnov test) of the differences is indicated above each
bar; p values less than or equal to 0.01 are highlighted in red. e, Normalized miRNA abundances of Cdcl3 (in three biological replicates). f, Cumulative
distributions (top) and significance (bottom; the red dotted line indicates p value of 0.01) of TE (left), RPF (middle) and RA (right) log2 fold changes
for mRNAs containing miR-C89 target sites exclusively within the CDS (green) or 3UTR (blue) (sample sizes 141 and 25, respectively). 5 UTR-exclusive
targets were omitted due to low sample size.

complying with the plant targeting rule in Chlamydomonas20. Finally, we tested the effect of miRNA abundance on TE, RPF and
Moreover, of these potential CDS cleavage site mRNAs, only 18/83 RA by focusing on the most abundant miRNA in our corresponding
were expressed in our data set, albeit at very low levels (Supplementary sRNA-Seq data sets: miR-C89 (Fig. 3e,f and Supplementary Fig. 5;
Fig.6C). We also investigated potential targets for expressed miRNA 5UTR and protein data excluded due to small sample size). miR-C89
where the base-pairing is between positions 2 and 15 (allowing one correlated with a larger shift in TE and RA than other miRNAs, con-
mismatch) and, similar to the plant-rule potential targets, there were sistent with the magnitude of the effect being influenced by miRNA
very few candidates (47 in total), of which only 31 are expressed abundance.
in our data set and the expression level for all 31 mRNAs is low From these findings, we conclude that, similar to metazoan
(Supplementary Fig. 6C). Thus, well expressed genes are unlikely to systems8,9, Chlamydomonas miRNA generally fine-tunes gene
be cleaved under steady-state conditions, consistent with the lack of expression through an effect on both RNA abundance and transla-
phenotype for both dcl3-1 and ago3-25 mutants. A recent degradome tion efficiency (Fig. 3). The global effect on translation efficiency
study is also consistent with there being minimal miRNA target site was significant although smaller than the effect on RNA abun-
cleavage in Chlamydomonas. The study involved miR-910, an miRNA dance (Fig.3a,b), as in metazoans9. Unlike metazoans, however, the
also expressed in our sample, that cleaved only two mRNAs following primary targets of miRNAs in Chlamydomonas are in the CDS
salt-stress29. The endogenous miRNA-mediated RNA downregulation instead of 3UTRs (Fig.3). This difference may reflect differences
by CDS-targeted miRNA is not, therefore, likely to be mainly through between Chlamydomonas and metazoans in the ways in which
target cleavage. miRNAs may influence elongating ribosomes.

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NATURe PlAnTs Letters
a dcl3-1

Cdcl3 6 C B

log2(RA fold change)

2
DCL3

0 rpL14
A A
2 Cre16.g675200

dcl3-1

6 B C
Cdcl3

2 1 0 1 2
dcl3-1 dcl3-1

log2(TE fold change)
Cdcl3 Cdcl3

b rpL14 c Cre16.g675200 d DCL3
1,500 10 350 1,836
400 352 310 1,292 1,873
200 6 30
8 5 25
300 150
1,000 6 4 20
200 100 3 15
4 2 10
500 50
100 2 1 5
0 0 0 0 0 0
29 442 238 2,883 612 12,830
rpL14 Cre16.g675200 DCL3
400 1,500 10 200 6 30
8 5 25
300 150
1,000 6 4 20
200 100 3 15
500 4
2 10
100 2 50
1 5
0 0 0 0 0 0
29 442 238 2,883 612 12,830

Hygromycin insertion site (nucleotide 10,193)

e ago325

6 C B
WT

4
log2(RA fold change)

0
A A
2

ago325 6 B

C
WT

2 1 0 1 2
ago325 ago3-25

log2(TE fold change)

WT WT

Fig. 4 | Effects of miRNAs on TE and RA. a, Correspondence between TE and RA fold changes between dcl3-1 and Cdcl3 for nuclear-encoded genes containing
miRNA target sites exclusively within the CDS (except DCL3, which was included as a marker). 80S, chloroplast and mitochondria ribosomal proteins are in
orange, green and red, respectively. b,c, Histograms of 5end positions of normalized RPF (coloured, left axis) and RNA-Seq (grey, right axis) 27-nucleotide
reads mapped to genes with high differential TE: ribosomal proteins rpL14 and Cre16.g675200. The top (green title) and bottom (red title) graphs are derived
from either Cdcl3 or dcl3-1, respectively. The coloured horizontal line indicates the riboSeqR de novo-defined ORF; positions of potential miRNA target sites
are annotated. d, Histogram of 5end positions of normalized RPF (coloured, left axis) and RNA-Seq (grey, right axis) 27-nucleotide reads mapped to DCL3
transcripts. The blue horizontal line indicates the CDS (nucleotides 61212,830). The schematic below the plot shows the domain organization of DCL3, which
contains two DEAD/DEAH box helicase domains (light and dark red boxes), a helicase domain (purple box), a proline-rich domain (orange box) and two
ribonuclease III domains a and b (light and dark green boxes, respectively). The thick grey line and the corresponding red arrow below indicate the hygromycin
insertion site (nucleotide 10,193). e, Correspondence between TE and RA fold changes between ago3-25 and the wild type CC-1883 for nuclear-encoded genes
containing miRNA target sites exclusively within the CDS. Nuclear-encoded 80S and chloroplast ribosomal proteins are in orange and green, respectively.
Mitochondrial ribosomal proteins are not shown due to the low level of detection in the data set.

miRNA and ribosomal protein TE and RA for all mRNAs with CDS-exclusive target sites (Fig.4).
Our finding that miRNA targeting in Chlamydomonas is Using the dcl3-1 mutation as a benchmark (log2(FC(TE))= 0.7
influenced by miRNA abundance and the number of target and log2(FC(RNA))= 1.18), individual RNAs that are
sites (Fig.3) implies that some mRNAs may be affected more negatively regulated by miRNAs would distribute in field A
than others. Therefore, to detect possible changes in individual of this figure if TE is affected (that is, log2(FC(TE))< 0.7,
mRNAs, we plotted the dcl3-1 versus Cdcl3 differences in TE yellow shaded area), field C if RA is affected but not TE (that

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Letters
is log2(FC(RA))< 1.18, 0.7 < log2(FC(TE))< 0.7, purple sites and the distribution of RPF or RNA reads for the mRNAs of
shaded area) and in field B if there was a double effect on
both TE and RA (log2(FC(RA))< 1.18, log2(FC(TE))< 0.7,
red shaded area). Corresponding positive regulation
would be indicated by distribution in fields A', B' and C'
respectively (Fig.4a).
The distribution of mRNA in this plot is consistent with a higher
degree of negative rather than positive regulation on a few mRNAs:
there were 32 and 16 targets in A and A' respectively, 3 and 0 in B
and B', and 15 and 3 in C and C'. From this analysis, we conclude
that there may be up to 32 mRNAs that are subject to strong transla-
tional regulation by miRNAs (from the A and B fields), 15 subject to
strong regulation of RNA abundance (from the B and Cdcl3 fields)
and 3 subject to strong regulation at both levels. The RNA-Seq and
RPF data for DCL3 mRNA and selected miRNA targets including
rpL14 and Cre16.g67520 from field A are presented in Fig.4ce.
To assess whether the mRNAs in field C could either be miRNA
targets or they could have DCL3 cleavage sites, we monitored
their level in ago3-25 and the wild type (Supplementary Fig.6D).
Repression of RNAs that are targeted by DCL3 would be relieved in
dcl3-1 but not ago3-25, whereas those that are targeted by miRNAs
would be depressed in both mutants.
The data are consistent with miRNA targeting for most of the field C
RNAs of Fig.4 because their repression was relieved in both mutants
although Cre15.g643503.t1.1 was an exception (perhaps related to it
having an unusually long CDS7,884 nucleotides, compared with
the average CDS length for expressed genes of 2,429 nucleotides;
Supplementary Fig.6D). We therefore conclude that the RA effect we
observe is genuinely directed by the miRNAAGO complex. Further,
to distinguish whether reduced expression in Cdcl3 relative to
dcl3-1 was a global effect or merely due to a small number of strongly
repressed genes (that is, fields A, B, C, A', B' and C' of Fig. 4a), we
repeated the analysis with the strongly repressed candidates excluded
and found a similar pattern of global mRNA repression as with all
mRNAs (Supplementary Fig. 7A). Similarly, with the targets of
miR-C89, the repression of TE or RA primarily results from small
changes in the expression of many genes (Supplementary Fig.7B).
It is striking that mRNAs subject to either strong translational or
RNA stability regulation (that is, fields A and C) are enriched with
those encoding RNA-interacting proteins (for example, transla-
tion, transcription and ribosomal RNA processing) (Supplementary
Table3). Of the mRNAs subject to translational regulation, a gene
ontology analysis revealed the enriched pathway of translation
and ribosome with the mRNAs for 80S ribosomal proteins being
particularly prominent (Fig.4a and Supplementary Table3). These
candidates also contribute to the outlier group for TE and RPF but
not RA in the cumulative distributions for transcripts with support-
ing proteomic data (Supplementary Fig.6B). Furthermore, the same
enrichment is also observed in the ago3-25 mutant (Fig. 4e and
Supplementary Fig.7C). However, we do not observe enrichment
for this pathway in previously published mammalian data sets9 of
miR-233-knockout cultured neutrophils compared with wild-type
cultured neutrophils, and HeLa cells after transfection with miR-1
or miR-155 (Supplementary Fig.8).
The enrichment of translation and ribosome function in fields A
and C of Fig.4a,e is specific for 80S ribosomal proteins; the nucleus-
encoded 70S ribosomal proteins for both chloroplasts and mitochon-
dria were an internal control and cluster around the zero-fold change
axis for both TE and RNA (Fig. 4a,e). It is likely therefore that the
specific effect for the 80S factors reflects the targeting specificity of
miRNAs in Chlamydomonas or that it is a compensatory mechanism
for the loss of a layer of regulation in the dcl3-1 and ago3-25 mutants.
It is possible that the distribution of ribosomes on the mRNA
would be affected by absence of miRNAs (see Fig.4b,c; for exam-
ple, rpL14 and Cre16.g675200). However, we did not observe any
significant correlation between the position of the miRNA target
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NATURe PlAnTs
fields A and C of Fig. 4a either individually or through a global
analysis of multiple RNAs. In contrast, in the mRNA for DCL3
there was an effect: the RPFs in the Cdcl3 sample extended to the
stop codon and the RNA-Seq reads covered the full length of the
mRNA, whereas in dcl3-1 the RPF and RNA-Seq data were more
sparse than in Cdcl3 and they stopped at the site of the mutagenic
hyg insert (Fig.4d and Supplementary Fig.3C). Clearly, from this
DCL3 analysis, the RPF and RNA-Seq data can reflect both the
quantitative and qualitative aspects of ribosome distribution and
RNA accumulation.
We hypothesized that CDS-targeting of the miRNAAGO com-
plex should result in road-blocking of elongating ribosomes, result-
ing in ribosome pile-up and/or drop-off of the 5and 3ends of
miRNA target sites respectively. However, we did not observe any
significant changes in RPF density around miRNA target sites,
indicating that the RNA-induced silencing complex (RISC) does
not induce ribosome pile-up within CDS regions. Presumably the
efficient RNA helicase activity of the ribosomes is able to overcome
the steric hindrance by the RISC in Chlamydomonas30,31. There may,
however, be a transient effect on ribosome translocation. Having
now identified these RNAs with the greatest effect on TE and RNA,
we will be able to explore the factors affecting the two modes of
RNA regulation and the conditions under which miRNAs have the
greatest effect on their mRNA targets.
Methods
Culturing and harvesting Chlamydomonas. Three independent fresh single
colonies of Chlamydomonas reinhardtii cells were sub-cultured as biological
triplicates. Cells where grown in 50ml Tris-acetate-phosphate (TAP) medium at
23C in baffled flasks on a rotatory shaker (21,100 g) under constant illumination
with white light (70E m2 s1) to mid-log phase (OD750 nm ~ 0.6), followed by
inoculation into 750ml TAP in 2l baffled flasks at OD750 nm=0.2. These were
cultured in the same conditions until mid-log phase prior to harvesting by
filtering off the media, after which the cell paste was immediately flash-frozen and
pulverized in liquid nitrogen with 5ml of pre-frozen buffer (20mM
Tris-Cl pH 7.5, 140mM KCl, 5mM MgCl2, 10g ml1 cycloheximide, 100g ml1
chloramphenicol, 0.05mM dithiothreitol (DTT), 0.5% NP40, 1% Triton X-100
and 5% sucrose). The frozen powder was gradually thawed on ice and clarified by
centrifugation for 30min at 4,194 g at 4C followed by adjustment of
A254 nm=10 before further treatment, or snap-frozen in liquid nitrogen and
stored at 80C. The extraction efficiency was monitored by polysome profiling
(Supplementary Fig.3F). The flash-freezing method was preferred as methods
involving pretreatment with translational inhibitors such a cycloheximide
or chloramphenicol can introduce various biases, in particular in artificially
enhancing the initiation peak of the profile32, which we also observed in
Chlamydomonas reinhardtii when we compared flash-freezing with cycloheximide
pretreatment (Supplementary Fig.3G).
Metabolic labelling and LCMS/MS. For metabolic labelling, ammonia chloride
(14N) was replaced with ammonia chloride15N (Cambridge Isotope Laboratories)
in the TAP media used to maintain dcl3-1. There were no obvious differences in
growth rates between algae maintained in 14N and 15N. dcl3-115N and Cdcl314N
were mixed equally prior to protein extraction via TCA/acetone precipitation
followed by resuspension in resuspension buffer (8M urea, 500mM NaCl, 10mM
Tris-Cl pH 8, 5mM DTT) and resolved in 1.5mm 10% bis-tris Novex Gel (Thermo
Fisher Scientific). The experiment was performed in biological triplicate.
One-dimensional gel bands (12 per lane) were transferred into a 96-well PCR
plate. The bands were cut into 1 mm2 pieces, de-stained, reduced (DTT), alkylated
(iodoacetamide) and subjected to enzymatic digestion with trypsin overnight
at 37C. After digestion, the supernatant was pipetted into a sample vial and
loaded onto an autosampler for automated liquid chromatographytandem mass
spectrometry (LCMS/MS) analysis.
All LCMS/MS experiments were performed using a Dionex Ultimate 3000
RSLC nanoUPLC (Thermo Fisher Scientific) system and a QExactive Orbitrap
mass spectrometer (Thermo Fisher Scientific). Separation of peptides was
performed by reverse-phase chromatography at a flow rate of 300nl min1 and
a Thermo Scientific reverse-phase nano Easy-spray column (Thermo Scientific
PepMap C18, 2m particle size, 100 pore size, 75m i.d. 50cm length).
Peptides were loaded onto a pre-column (Thermo Scientific PepMap 100 C18, 5m
particle size, 100 pore size, 300m i.d. 5 mm length) from the Ultimate 3000
autosampler with 0.1% formic acid for 3min at a flow rate of 10l min1. After this
period, the column valve was switched to allow elution of peptides from the
pre-column onto the analytical column. Solvent A was water+0.1% formic acid
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NATURe PlAnTs
and solvent B was 80% acetonitrile, 20% water+0.1% formic acid. The linear
gradient employed was 240% B in 30min (total run time including a high organic
wash step and re-equilibration was 60min).
The LC eluant was sprayed into the mass spectrometer by means of an
Easy-Spray source (Thermo Fisher Scientific). All m/z values of eluting ions
were measured in an Orbitrap mass analyser, set at a resolution of 70,000 and
was scanned between m/z 380 and 1,500. Data-dependent scans (Top 20) were
employed to automatically isolate and generate fragment ions by higher energy
collisional dissociation (HCD, normalized collision energy: 25%) in the HCD
collision cell and measurement of the resulting fragment ions was performed in
the Orbitrap analyser, set at a resolution of 17,500. Singly charged ions and ions
with unassigned charge states were excluded from being selected for MS/MS and a
dynamic exclusion window of 20s was employed.
Protein identification and relative quantification. Data were recorded using
calibur software version 2.1 (Thermo Fisher Scientific). Files were converted
from .raw to .mzXML using MSConvert and then .mzXML files to .mgf using
the in-house software iSPY33,34. The mgf files were submitted to the Mascot
search algorithm(Matrix Science, London UK) and searched against the NCBI
Chlamydomonas reinhardtii database (14491 sequences; 6575547 residues).
The following parameters were employed: carbamidomethyl as a fixed
modification, and oxidation on methionine residues and phosphorylation on
serine, threonine and tyrosine residues as variable modifications; 20 ppm for
peptide tolerance, 0.1Da of MS/MS tolerance; a maximum of two missed cleavages,
peptide charges of+2, +3 or +4; and selection of a decoy database. Mascot.dat
output files were imported into iSPY for 14N/15N quantification and analysed
through Percolator for improved identification35. The 14N and 15N peptide isotopic
peaks from the MS1 data set were used to compare the theoretical mass difference
between the heavy and light peptides, and the typical isotopic distribution patterns.
Only unique peptides with a posterior error probability of0.05 were considered
for further analysis. Spectra were merged into peptides and proteins based on their
median intensity in MS1, meaning the more intense the signal of the spectrum, the
more weight it added to quantification. The statistical programming environment
R was used to process iSPY output files to check for the 15N incorporation rate
and to confirm that the data were normally distributed. After normalization, only
peptides detected in at least two biological replicates, with a fold change>1.5 and
a p value0.05 were considered for further analysis. Relative protein expression
values were computed as (ProteinCdcl3/Proteindcl3-1) using the average of the
triplicates for all follow-up analysis.
Nuclease footprinting. Lysates (200l) were slowly thawed on ice and treated
with 6,000 units RNase I (Thermo Fisher Scientific) in a thermo-mixer at 28C,
400r.p.m. for 30min. The reaction was stopped by mixing the digest reaction with
120 units of SUPERase-In RNase inhibitor (Thermo Fisher Scientific) followed
by centrifugation for 2min at 21,100 g at 4C to further clarify any remaining
debris. The supernatant was layered onto a 1M sucrose cushion prepared in
Chlamydomonas polysome buffer, and RNAs were purified as described in
Ingolia et al.36. Polysome integrity for the lysate and digestion conditions were
assessed via polysome profiling (Supplementary Fig.3F).
Letters
Differential analyses for the mouse data in Guo et al. 20109 were obtained from the
Gene expression Omnibus in NCBI (accession: GSE220001 and GSE21992).
Target prediction. Target prediction was done using TargetScan26 using the
same transcriptome input as for the ribosome profiling analysis. As there are no
conserved sites available due to the lack of miRNA data from the green algae
phylum, we could not calculate context and scores; thus, we utilized only the part
of the software to detect all possible miRNA target sites. Further, as the efficacies
between octonucleotide-A1 and octonucleotide-m8 sites are similar, we combined
both types of target site in the octonucleotide prediction, similarly to Guo et al.
2010 and Agarwal et al. 20159,26. Target prediction based on the plant rule was
performed via TAPIR38.
The list of miRNAs used was based on the 19 DCL3-dependent miRNAs
expressed according to the sRNA data, where the average number of reads within
the complement is greater than 400 and the average ratio of complement to
dcl3-1 reads is greater than 150. The selected DCL3-dependent miRNAs used are:
chromosome_5_3227666_3227753_+ (miR-C89),
chromosome_6_6776108_6776193_+ (miR-cluster20399),
chromosome_13_2001067_2001197_ (miR-cluster 7085),
chromosome_10_3399870_3399999_ (miR9897),
chromosome_13_3152367_3152452_ (miR-C112),
chromosome_6_3067368_3067456_+ (miR1162),
chromosome_12_6402226_6402307_ (miR1157),
chromosome_9_6365928_6366014_ (miR912),
chromosome_7_4386252_4386309_,
chromosome_17_6144120_6144204_+ (miR-cluster12551),
chromosome_1_7070552_7070605_, chromosome_16_185088_185174_
(miR1169), chromosome_2_8349161_8349264_+,
chromosome_2_9129508_9129593_ (miR-cluster14712),
chromosome_7_5926395_5926482_+ (miR-C59),
chromosome_14_3218783_3218866_ (miR910),
chromosome_6_7063792_7063881_ (miR1152),
chromosome_4_3100624_3100751_+ (miR1153) and
chromosome_1_5106349_5106475_+(miR-C82). The miRNA precursor sequence
used for mapping was based on Valli et al. (2016). Only octonucleotide sites were
utilized, and octonucleotide complementarity was verified via extraction of target
sites followed by miRNA complementarity assessment using the Vienna RNA
package program RNAduplex. The level of 3complementarity was similarly
investigated where the fragment containing nucleotides 9 to 21 of the target site
3of the seed region was extracted and the level of complementarity was assessed
with RNAduplex.
Data and code availability. All raw sequencing data are deposited in Arrayexpress
with accessions E-MTAB-3852 and E-MTAG-3851. All code used for statistical
analysis is available on request.
Received: 14 April 2017; Accepted: 31 August 2017;
Published: xx xx xxxx

Ribosome profiling and RNA-Seq. The methodologies were largely based

on the protocols of Ingolia et al. and Guo et al.9,36 with the following
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modifications: mRNA for corresponding RNA-Seq was enriched by removal of
rRNA using the ribo-zero kit (plant seed and root kit); RNA-Seq size selection
was in parallel with ribosome profiling (that is, between 26 and 34 nucleotides);
and for ribosome profiling, ribosomal RNA contamination was removed
by two rounds of treatment with duplex-specific nuclease for 30min as
described in Chung et al. 201524.
Preparation for sRNA libraries. Small RNAs from total RNA samples used for
RNA-Seq were size excluded in 15% TBU gel for miRNA enrichment
(Thermos Scientific). The sRNAs were further prepared according to the
NEXTflex small RNA-Seq kit v2 (Bio Scientific), followed by sequencing on the
NextSeq500 platform.
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considered only if they had: at least ten normalized RPF and ten normalized RNA
counts; and the sum of all RPF or RNA counts over the three biological replicates
for both dcl3-1 and its complement combined is at least 200. Normalization
was based on BaysSeq output37. Cumulative distributions for TE, RPF and RA
fold changes were calculated on the basis of the average of all three replicates.
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Acknowledgements
We thank J. Barlow for technical assistance and medium preparation; T.J. Hardcastle
and B. Santos for technical bioinformatic support; A. Valli for providing the silencing
mutants, and A. Molnar and A.E. Firth for discussions. This work was supported by
a Balzan Prize award and the European Research Council Advanced Investigator Grant
ERC-2013-AdG 340642 TRIBE(D.C.B). B.Y.-W.C. was supported by an EMBO long-
term postdoctoral fellowship and a Sir Henry Wellcome Fellowship [096082].
D.C.B. is the Royal Society Edward Penley Abraham Research Professor.
Author contributions
B.Y.-W.C. and D.C.B. conceived and designed the research. B.Y.-W.C performed the
experiments and analysed the data. M.J.D., A.J.G. and J.H. performed all the LCMS/MS
sample processing and iSPY analysis. B.Y.-W.C. and D.C.B. wrote the manuscript.
Competing interests
The authors declare no competing financial interests.
Additional information
Supplementary information is available for this paper at doi:10.1038/s41477-017-0024-6.
Reprints and permissions information is available at www.nature.com/reprints.
Correspondence and requests for materials should be addressed to B.Y.-W.C. or D.B.
Publishers note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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